Purification and characterization of glycerol kinase from Trypanosoma brucei.
Glycerol kinase (EC 2.7.1.30)(GK) from the glycosomes of Trypanosoma brucei has been purified and its kinetic properties have been examined. It has a molecular weight of approximately 53,000 and exists in solution as a monomer. This GK has a broad pH optimum, with equal activity between pH 7 and 9.5. Its catalytic mechanism appears to be random bi bi, with some cooperativity in substrate binding at high pH. The apparent Michaelis constants are: Kglycerol = 0.26 +/- 0.02 mM and KATP = 0.19 +/- 0.02 mM at pH 7.4, and Kglycerol = 0.17 +/- 0.03 mM and KATP = 0.26 +/- 0.02 mM at pH 9.0. Glycerol-3-phosphate (G3P) up to 10 mM displays virtually no product inhibition of the forward reaction, but ADP is a weak inhibitor, competitive with ATP and uncompetitive with glycerol. The forward reaction is catalyzed very efficiently in vitro, but the reverse reaction proceeds at an extremely low rate, consistent with its unfavorable delta G. Under anaerobic conditions T. brucei GK is thought to convert ADP and G3P to ATP and glycerol rapidly inside the intact glycosome, where it is tightly coupled to the other glycosomal enzymes. Our kinetic analyses suggest that GK may not rely on any unusual intrinsic properties to catalyze this reverse reaction: rather, the unusually high intraglycosomal concentrations of G3P and ADP, and the presence of efficient ATP traps, may drive this reaction by mass action.
Krakow
J L
JL
Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco 94143.
Wang
C C
CC
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D005991:Glycerol Kinase / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D005991:Glycerol Kinase